Metabolic Solutions offers project
design assistance and a mass spectrometry service
to help researchers determine energy expenditure using
the doubly labeled water stable isotope method.
Stable isotopes of hydrogen (deuterium oxide) and
oxygen (oxygen 18) are routinely used to measure energy
expenditure in free-living humans. The doubly labeled
water method using these isotopes is a form of indirect
calorimetry that has been extensively validated in
animals and humans. The method is completely safe,
requires only periodic sampling of body fluids, is
non-restrictive, and is ideally suited for measurement
of energy expenditure in free-living or hospitalized
patients.
A typical energy expenditure study protocol using
the doubly labeled water method starts with a baseline
urine collection to determine pre-dose values for
the hydrogen and oxygen isotopes. The subject is given
a single oral bolus dose of heavy water (2H218O).
Generally, adults are given a dose consisting of 0.15
g H218O/kg body weight and 0.06
g 2H2O/kg body weight. Children
and neonates are given higher doses per kilogram due
to their faster water turnover rates.
Background
The development of the doubly labeled water method
for energy expenditure originated from a study by
Lifson et al. in 1949. Using the stable isotope of
oxygen, Lifson et al. administered 18O-labeled
water to animals and showed that the 18O-label
appeared in expired CO2. This demonstrated
that expired CO2 was derived from body
water.
Further experiments by Lifson, Gordon and McClintock
showed that total daily CO2 production
could be measured from the differential elimination
of water labeled with stable isotopes of hydrogen
and oxygen. After the administration of doubly labeled
water (2H218O), the
labeled hydrogen (2H2) would
be eliminated as water (2H2O),
corresponding to water output, whereas the oxygen
isotope would be eliminated as water (H218O)
and as expired carbon dioxide (C18O2).
By measuring the difference between the elimination
rates of labeled oxygen and hydrogen, the carbon dioxide
production rate can be calculated. The carbon dioxide
production rate is converted into energy expenditure
by knowing the respiratory quotient (RQ) of the food
ingested during the observation period.
Lifson Model
The model of Lifson is based on the total body water
pool (N) is a homogeneous compartment that remains
constant during observation. Further assumptions
implicit in the model are that the tracer isotopes
of hydrogen and oxygen exit the body only as water
and carbon dioxide and that dietary and atmospheric
sources of water and oxygen do not change the background
levels of isotopes. The basic mathematical equation
relating carbon dioxide production to the isotope
elimination rates is given in equation 1:
rCO2 = (N/2) (k18 - k2)
(1)
where N is the total body water pool, k18
is the rate of disappearance of 18O and
k2 is the rate of 2H disappearance.
The practical application of the method required the
incorporation of isotopic fractionation factors to
account for fractionation (non-equal equilibration)
of the isotopes of water and carbon dioxide during
changes in state. It had been recognized that
isotopically labeled water and carbon dioxide would
leave the body at different rates depending on its
chemical state, either gas or liquid. Measured
isotope fractionation factors for deuterium and 18O
indicate that breath, water, non-sweat water vapor
and expired CO2 are isotopically fractionated
relative to body water. With this correction,
the equation describing the model becomes:
rCO2 = (N/2 f3) (k18
- k2) - rH2OG (f2
- f1)/2 f3
(2)
where f1 is the deuterium fractionation factor between
water and water vapor, f2 is the 18O fractionation
factor between water and water vapor, f3 is the 18O
fractionation between water and carbon dioxide and
rH2OG is the rate of water loss
via isotopically fractionated routs.
Appropriate choices for the fractionation factors
in infants and adults has been the subject of numerous
papers. The isotope fractionation factors currently
used are:
f1 = 0.941 2H2O (gas)
/ 2H2O (liquid)
f2 = 0.992 H218O (gas)
/ H218O (liquid)
f3 = 1.039 C18O2 (gas)
/ H218O (liquid)
Model Assumptions
The doubly label water model developed
by Lifson incorporated many assumptions about the water
pool, water and CO2 flux, and the isotope
exchanges with the body pools. These assumptions
are estimates that have been shown to be reasonable
in much testing.
Constant Water Pool
The model assumes a constant water pool volume during
the metabolic period. The pool volume will change
with eating and drinking but over a 24 hour period, these
changes are quantitatively insignificant with respect
to the total pool size. However, the application
of the method to a growing premature infant, which increases
total body water by 20% in the period of 1 week, requires
a linear growth model to calculate the water pool sizes.
Two point and multipoint regression models have dealt
with the steady-state kinetics of the water and CO2
fluxes. The water and CO2 fluxes do change
episodically. However, these models have shown that
they can estimate the average flux over the metabolic
period to a high degree of accuracy.
Exchange of Isotopes with Water and CO2
Pools
The most important controversy of the doubly labeled
water method has been the assumption that the isotopes
are only exchanged with the body water and CO2
pools. It is now well known that the hydrogen
dilution space, estimated from the extrapolation of
the elimination curves back to time zero, has been observed
to be between 2 and 6% larger than the body water pool
as determined by desiccation. Furthermore, the
hydrogen dilution space is consistently larger than
the oxygen dilution space. This implies that the
hydrogen isotope exchanges with other pools in the body.
It has been suggested that the hydrogen exchanges with
acidic amino acids in proteins. The oxygen dilution
space appears to overestimate the body water pools by
about 1%. The oxygen isotope can exchange with
inorganic compounds in the body.
There is not total agreement on how to correct the estimates
of the water and CO2 fluxes due to these overestimates.
Schoeller et al. has used an average relationship, based
on all his human data, between the isotope dilution spaces
and the total body water pool size: hydrogen dilution
space/1.04 = oxygen dilution space/1.01 = total body water.
Roberts, Coward and Lucas have used individually determined
dilution spaces in their model. It is very important
to determine the dilution spaces accurately because small
errors are magnified 3- to 5-fold in the calculation of
the CO2 production rate.
Validation of Lifson Model
Inspite of the controversies about the values to use
for the water pool size, the doubly labeled water method
has been validated in humans against continuous respiratory
gas exchange measurements. Schoeller et al. and
Westerterp et al. have used the two point method for their
validations. Schoeller has completed validations
in 33 subjects ranging fro adults, to infants, to total
parenteral nutrition patients. The mean difference
from the respiratory gas exchange method was 0.6% with
a standard deviation of 6%. Other laboratories have
validated the doubly labeled water method using the multipoint
method and achieved roughly the same level of precision.
Typical Energy Expenditure Protocol
Theoretically, any body fluid can be sampled for measurement
of the water isotopes. Thus, blood, saliva and
urine can be sampled. However, urine samples
are most often the choice used by investigators because
of the ease of collection and availability of fluid.
The total amount of fluid necessary for both analyses
is about 2.5 ml per time point. Therefore, we
will discuss urine collections in our methodology.
Protocol
Following an overnight fast (about 8 hours), urine specimens
are collected before the administration of the isotope
dose. This will serve as the baseline isotope measurement.
A double-labeled dose of water is orally administered
to each subject. A mixed 2H218O
dose containing 0.15 gm/kg body weight of H218O,
99 atom % excess 18O, (or 1.5 gm/kg body weight
of H218O, 10 atom % excess 18O),
and 0.06 gm/kg BW 2H2),>99 atom
% excess, is given orally and then followed up with 100
ml of tap water. The first urine collection is four
hours following the dose. This is used to determine
the isotope dilution space and total body water.
The next urine collection is 24 hours for the isotope
dose. Thereafter, a minimum of two samples, at the
beginning and end of the study period, are necessary to
determine energy expenditure during the study period.
However, we recommend at least three or greater urine
collections per week for most accurate results.
Although many have argued that the two point method gives
equal accuracy, our experience has suggested that a linear
regression of more that two points gives best accuracy.
Urine Samples
All urine samples should be collected in non-acidified
plastic bottles. The urine should be aliquoted
immediately into smaller plastic tubes (about 5 ml urine)
and stored frozen (-20 °C) until analysis. Save
two or three aliquots. It is preferable that plastic
tubes that have been specifically designed for storage
at low temperatures be used.
Diets The respiratory quotient of the diet is used in
the Weir equation for determining energy expenditure.
Black et al. provides respiratory quotients from food
composition (Human Nutr. Clin. Nutr. 40C:381-391, 1986).
Alternatively, respiratory quotients can be measured directly
by respiratory gas exchange measurements, or by maintaining
a complete diet record for the study period.
Nitrogen Balance
Nitrogen balance during the study period will be required
for determining energy expenditure using the Weir equation.
A reference value of nitrogen excretion can be used instead
of laboratory measurement.
Analytical
Methods
All isotope measurements are made using an isotope ratio
mass spectrometer. The deuterium measurements use
equilibration of the water with hydrogen gas. The
18O measurements determine C18O2
using H2O-CO2 equilibration system.
Calculation of Energy Expenditure
Measurements
The delta deuterium and oxygen-18 values for the pre-dose
(dpre) and post-dose samples (dpost)
are determined. The doubly labeled dose is diluted
with tap water. The amount of dose diluted and
water used is recorded. The deuterium and oxygen-18
content of the tap water (dtap) and diluted
dose (ddose) are measured.
Treatment of Mass Spectrometric Data The unprocessed
mass spectrometric data is expressed as a fraction of
the initial dose given as suggested by the consensus
report by the International: Dietary Energy Consultancy
Group at the 1990 Vienna Austria Meeting (AM Prentice).
The Doubly-Labeled Water Method for Measuring Energy
Expenditure: Technical Recommendations for Use
in Humans. Vienna: Nahres-4, International Atomic
Energy Agency; 1990. This is achieved using the
formula:
X = ((dpost - dpre) / (ddose
- dtap)) x (18.02a / WA)
where W = Amount of water (grams) used to dilute the
dose, A = Amount of dose (grams) administered to subject,
a = amount of dose (grams) diluted for analysis.
Linear regression is used to calculate the slope and intercept
of the linear relationship between the time in days and
the normalized data for each isotope. The pool sizes
ND and NO are derived as the reciprocal
of the intercept (or plateau value). The intercept
of the regression line is the ratio of the pool size spaces
ND/NO. The multipoint data
is plotted to inspect for any outliers. Any outliers
are re-analyzed. The rate constants kD
and kO are represented by the slope of the
regression line. ND/NO ratios
lying outside the range of 1.015 and 1.06 are treated
as suspect and samples will be re-analyzed.
Calculation of Daily CO2 Production
The mean daily CO2 production (rCO2, mol/day) is calculated
according to the revised equations of Speakman, Nair
and Goran (Am. J. Physiol. 264: E912-E917, 1993):
rCO2 = (N/2.196) x (kO - 1.0427kD)
where N = [(NO) + (ND/1.0427)]/2.
Calculation of Energy ExpenditureThe estimate of energy
expenditure is calculated from the carbon dioxide production
assuming 127.5 kcal/mol carbon dioxide (a typical Western
diet will produce a respiratory quotient of 0.85, with
15% of energy from protein oxidation, as suggested by
Elia in the IDECG consensus report, Vienna 1990 Meeting).
The use of a general value for the conversion of CO2
to energy expenditure for a "western" type diet
was found to predict to within 5% the energy expenditure
of 63 randomly-selected individuals.
Published
Energy Expenditure Studies Analyzed by Metabolic
1. Hinchcliff, K.W., Reinhart, G.A., Burr, J.R., Schreir,
C.J., Swenson, R.A. Metabolizable energy intake and
sustained energy expenditure of Alaskan sled dogs during
heavy exertion in the cold. Am J Vet Res, 58(12):1457-1462,
1997.
"To measure energy expenditures
of Alaskan sled dogs at rest and during racing under
frigid conditions, using the doubly labeled water technique."
2. Coup, R.N., Pekins, P.J. Field metabolic rate
of wild turkeys in winter. Can. J. Zool. 77:1075-1082,
1999.
"We investigated the winter bioenergetics
of eastern wild turkeys (Meleagris gallopavo sylvestris)
by measuring standard metabolic rate (SMR) and existence
metabolism (EM) of captive turkeys and field metabolic
rate (FMR) of free-ranging turkeys."
3. Perks, S.M., Roemmich, J.N., Sandow-Pajewski, M.,
Clark, P.A., Thomas, E., Weltman, A., Patrie, J., Rogol,
A.D. Alterations in growth and body composition during
puberty. IV. Energy intake estimated by the Youth-Adolescent
Food-Frequency Questionnaire: validation by the doubly
labeled water method. Am J Clin Nutr 72:1455-1460, 2000.
"Our objective was to validate energy intake
estimated by the Youth-Adolescent Food-Frequency Questionnaire
(YAQ) against the criterion total energy expenditure
(TEE) by doubly labeled water (DLW)."
4. Costa, D.P., Gales, N.J., Foraging energetics and
diving behavior of lactating New Zealand sea lions,
Phocartos Hookeri. Journal of Experimental Biology,
203:3655-3665, 2000.
"We measured the
metabolic rate, water turnover and diving behavior of
12 lactating New Zealand sea lions at Sandy Bay, Enderby
Island, Auckland Islands Group, New Zealand (50°30's,166°17'E),
during January and February 1997 when their pups were
between 1 and 2 months old."
5. Shaffer, S.A., Costa, D.P., Weimerskirch, H.
Comparison of methods for evaluating energy expenditure
of incubating wandering albatrosses. Physiol Biocem
Zool, 74(6): 823-831, 2001.
"Measurements
of incubation energetics can vary depending on the method
used to measure metabolism of an incubating bird.
Therefore, we evaluated the energy expenditure of six
male and four female wandering albatrosses (Diomedea
exulans Linnaeus) using doubly labeled water (DLW),
the rate of mass loss, and estimates of metabolic water
production derived from water influx rate (WIR)."
6. Conway, J.M., Seale, J.L., Jacobs, D.R. Jr., Irwin,
M.L., Ainsworth, B.E. Comparison of energy expenditure
estimates from doubly labeled water, a physical activity
questionnaire, and physical activity records. Am J Clin
Nutr 75:519-525, 2002.
"We compared energy expenditure (EE) as estimated
by indirect methods (physical activity records and recall
questionnaires) used in epidemiological studies with
EE obtained from doubly labeled water (EEDLW)
in free-living men."
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